CN100538339C - The method of synchronous detecting lily mottle virus, lily asymptomatic virus and cucumber mosaic virus - Google Patents
The method of synchronous detecting lily mottle virus, lily asymptomatic virus and cucumber mosaic virus Download PDFInfo
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Abstract
The invention provides the method for a kind of synchronous detecting lily mottle virus (LMoV), lily asymptomatic virus (LSV) and cucumber mosaic virus (CMV), platymiscium protection field.Its scheme is, by extracting steps such as total RNA, multiple RT-PCR and electrophoresis detection, 1～3 kind in LMoV, the LSV that under same reaction tube and same thermal cycle conditions lily is infected and three kinds of viruses of CMV is increased, and separates and detect by gel electrophoresis.This method mainly is to have realized three kinds of viral synchronous detection except quick, special, the sensitive advantage that has common RT-PCR and have, and has reduced cost.
Description
Technical field:
The present invention relates to lily mottle virus (LMoV), lily asymptomatic virus (LSV) and three kinds of main viral multiplex RT-PCR methods of cucumber mosaic virus (CMV) that a kind of synchronous detection infects lily, platymiscium protection field.
Background technology:
Lily be integrate view and admire, eat, medicinal important industrial crops, especially flower lily be bright-colored, elegant, and has unique fragrance, become one of most important ornamental flower in the world, has very high economic worth.In recent years, along with improving constantly of living standards of the people, to Fresh Cutting flower particularly the demand of lily constantly increase, the planting scale of lily enlarges day by day.Yet because the nonstandard mode of production and long-term vegetative propagation make constantly accumulation of virus, the virus disease of lily is on the rise, and greatly reduces the yield and quality of lily, causes enormous economic loss for lily plant husbandry.It is reported that the virus that infects lily has kind more than 10, it is wide to distribute, harm is big, serious lily mottle virus (LMoV), lily asymptomatic virus (LSV) and the cucumber mosaic virus (CMV) etc. of mainly containing cause harm, and these three kinds of viral Chang Fuhe infect, cause symptoms such as blade floral leaf, necrosis, deformity and the broken look of flower, seriously reduced commodity and the sight of lily.Usually the infection rate of lily virus is at 30-40%, have up to 100%, the harm that causes to lily is bigger.Up to the present, still the special effect agent that does not have the treatment virus disease in the world, therefore lily virus detects especially to three kinds of main viral detection and monitorings, having important and practical meanings for identification virus disease and the spread and epidemic that prevents virus, is to produce to go up one of important measures of the lily virus disease being carried out the comprehensive regulation.
Usually the method that detects lily virus has phyto-indicator method, electron microscopy, enzyme linked immunological adsorption technology and Protocols in Molecular Biology etc.Comparatively speaking, though the phyto-indicator method is simple, sense cycle is long, the shortlyest also takes 10～20 days, and sensitivity is very low, also is subjected to the influence of aspects such as season, environment and viral character, so seldom has been used for the detection of lily virus.The appearance of immuno-electron microscope has promoted the application of electron microscopy in the lily virus context of detection greatly, but electron microscope costs an arm and a leg, and the sample making technology complexity is difficult for grasping, and is not suitable as the commonsense method that lily virus detects.The method of the detection lily virus that is most widely used at present is enzyme linked immunosorbent assay (ELISA) and Protocols in Molecular Biology.Than phyto-indicator method and electron microscopy, ELISA method sense cycle is shorter, and is highly sensitive, can detect the disease of nanogram (ng) level level.Protocols in Molecular Biology, particularly the RT-PCR technology is not only simple to operate, and specificity, sensitivity can detect the virus that flies gram (fg) level level than ELISA method height.But the method for ELISA method and common RT-PCR can only detect a kind of virus at every turn, for the compound situation that infects lily of multiple virus, then need detect separately at every kind of virus, both loses time, and has strengthened the input that detects cost again.In addition, all need to design the upstream and downstream primer respectively for every kind of virus in the common RT-PCR amplification procedure, cause detecting cost and raise, and improved the occurrence probability of non-specific detection.
Summary of the invention:
The present invention has overcome classic method and has detected the compound deficiency that infects lily of multiple virus, provide a kind of synchronously, fast, the LMoV, the LSV that take place on sensitive, the special detection lily and the method for three kinds of viruses of CMV.
Step of the present invention is:
1) design primer:
(a) design detects the downstream universal primer LMSV of LMoV, two kinds of viruses of LSV:
LMSV 5-GATGGCTGACTGGATCCTTTTTTTTTTTTTTTTV-3 V=A, C or G
(b) design detects the upstream primer of LMoV, LSV, and primer sequence is as follows:
(c) design detects the upstream and downstream primer of CMV, and primer sequence is as follows:
(d) determine every pair of primer amplification clip size:
2) total RNA extracts:
(a) take by weighing lily blade 50mg, put into mortar, add 1mL Trizol reagent and grind, lapping liquid is poured in the 1.5mL centrifuge tube;
(b) add 400 μ L chloroforms, leave standstill 5min behind the mixing;
(c) 12, the centrifugal 10min of 000rpm;
(d) supernatant moves in the new 1.5mL centrifuge tube, adds isopyknic isopropyl alcohol, leaves standstill 5min behind the mixing;
(e) 12, the centrifugal 10min of 000rpm;
(f) abandon supernatant, precipitation is washed with 1mL70% ethanol, dry 10min;
(g) precipitation is handled aqueous suspension with 40 μ L DEPC, and-20 ℃ of preservations are standby;
3)RT-PCR：
(a) RNA sex change: RNA3 μ L, LMSV1 μ L, CMV1 μ L and 7.5 μ L ddH
2The O mixing, 70 ℃ of insulation 10min, rapid afterwards ice bath;
(b) reverse transcription: in aforementioned tube, add reagent by following system, the vibration mixing, room temperature is placed 10min, and 42 ℃ of insulation 1hr obtain cDNA;
(c) PCR: get a new PCR pipe, the according to the form below system adds each reagent:
The PCR program is set is: 94 ℃ of 4min, 94 ℃ of 1min, 53 ℃～60 ℃ 1min, 72 ℃ of 2min, 25～40 circulations of increasing, 4 ℃ of preservations behind 72 ℃ of extension 10min;
4) electrophoresis detection: the Ago-Gel of preparation 0.8%～2%, in 0.5 * tbe buffer pendular ring border, 150V voltage stabilizing electrophoresis 90min takes out gel afterwards and is immersed in the 10min that dyes in the 0.5 μ g/mL ethidium bromide, observes under uviol lamp then and the record result;
5) testing result analysis: having that it's too late size is determined the viral species that lily infects according to amplified band on the running gel; By observing, be the nucleic acid fragment of 1153bp if having size in the gel, then contain LMoV in the interpret sample; If having size in the gel is the nucleic acid fragment of 736bp, then contain LSV in the interpret sample; If having size in the gel is the nucleic acid band of 590bp, then contain CMV in the interpret sample.
Wherein, described PCR annealing temperature is 53 ℃～60 ℃, also can be 56 ℃～58 ℃; Described PCR circulation can be 25～40 circulations, also can be 26～29 circulations; Described agarose concentration can be 0.8%～2%, also can be 0.9%～1.2%.
The invention has the beneficial effects as follows and to save time and cost.Because 3 ' end of LMoV and LSV sequence all has a poly (A) structure, the primer that therefore can design a band Oligo d (T) in view of the above is as detecting the shared downstream universal primer of LMoV and LSV simultaneously, reduced the quantity of primer, and greatly reduce interaction between primer, and increased the specificity that detects.In addition, the present invention has overcome the limitation that traditional RT-PCR one individual system once can only detect a kind of virus, can detect three kinds of viruses simultaneously in an individual system, compares with traditional RT-PCR, can save 2/3rds detection time and reagent cost at most.
Description of drawings:
Fig. 1 is the electrophoresis detection figure of lily mottle virus, lily asymptomatic virus and cucumber mosaic virus.
Embodiment:
Example one:
With known MOI the lily plant of LMoV, LSV and three kinds of viruses of CMV be material, with the negative contrast of healthy lily plant, detect with this method.
1) design primer:
(a) design detects the downstream universal primer LMSV of LMoV, two kinds of viruses of LSV:
LMSV 5-GATGGCTGACTGGATCCTTTTTTTTTTTTTTTTV-3 V=A, C or G
(b) design detects the upstream primer of LMoV, LSV, and primer sequence is as follows:
(c) design detects the upstream and downstream primer of CMV, and primer sequence is as follows:
(d) determine every pair of primer amplification clip size:
2) total RNA extracts:
(a) take by weighing lily blade 50mg, put into mortar, add 1mL Trizol reagent and grind, lapping liquid is poured in the 1.5mL centrifuge tube;
(b) add 400 μ L chloroforms, leave standstill 5min behind the mixing;
(c) 12, the centrifugal 10min of 000rpm;
(d) supernatant moves in the new 1.5mL centrifuge tube, adds isopyknic isopropyl alcohol, leaves standstill 5min behind the mixing;
(e) 12, the centrifugal 10min of 000rpm;
(f) abandon supernatant, precipitation is washed with 1mL 70% ethanol, dry 10min;
(g) precipitation is handled aqueous suspension with 40 μ LDEPC, and-20 ℃ of preservations are standby;
3)RT-PCR：
(a) RNA sex change: RNA 3 μ L, LMSV 1 μ L, CMV 1 μ L and 7.5 μ L ddH
2The O mixing, 70 ℃ of insulation 10min, rapid afterwards ice bath;
(b) reverse transcription: in aforementioned tube, add reagent by following system, the vibration mixing, room temperature is placed 10min, and 42 ℃ of insulation 1hr obtain cDNA;
(a) PCR: get a new PCR pipe, the according to the form below system adds each reagent:
The PCR program is set is: 94 ℃ of 4min, 94 ℃ of 1min, 58 ℃ of 1min, 72 ℃ of 2min, 29 circulations of increasing, 4 ℃ of preservations behind 72 ℃ of extension 10min;
4) electrophoresis detection: the Ago-Gel of preparation 1.2%, in 0.5 * tbe buffer pendular ring border, 150V voltage stabilizing electrophoresis 90min takes out gel afterwards and is immersed in the 10min that dyes in the 0.5 μ g/mL ethidium bromide, observes under uviol lamp then and the record result;
5) electrophoresis detection result: infected in the lily blade of LMoV, LSV and three kinds of viruses of CMV having detected three nucleic acid bands of 1153bp, 736bp and 590bp, but illustrated by the present invention's synchronous detection and go out LMoV, LSV and three kinds of viruses of CMV; Do not detect any band in the normal healthy controls, illustrate that not contain these three kinds of viruses any.
Example two:
Detect with the known lily plant that has infected LMoV, LSV or CMV respectively, and with the negative contrast of healthy lily.
1) design primer:
(a) design detects the downstream universal primer LMSV of LMoV, two kinds of viruses of LSV:
LMSV 5-GATGGCTGACTGGATCCTTTTTTTTTTTTTTTTV-3 V=A, C or G
(b) design detects the upstream primer of LMoV, LSV, and primer sequence is as follows:
(c) design detects the upstream and downstream primer of CMV, and primer sequence is as follows:
(d) determine every pair of primer amplification clip size:
2) total RNA extracts:
(a) take by weighing lily blade 50mg, put into mortar, add 1mLTrizol reagent and grind, lapping liquid is poured in the 1.5mL centrifuge tube;
(b) add 400 μ L chloroforms, leave standstill 5min behind the mixing;
(c) 12, the centrifugal 10min of 000rpm;
(d) supernatant moves in the new 1.5mL centrifuge tube, adds isopyknic isopropyl alcohol, leaves standstill 5min behind the mixing;
(e) 12, the centrifugal 10min of 000rpm;
(f) abandon supernatant, precipitation is washed with 1mL 70% ethanol, dry 10min;
(g) precipitation is handled aqueous suspension with 40 μ L DEPC, and-20 ℃ of preservations are standby;
3)RT-PCR：
(a) RNA sex change: RNA 3 μ L, LMSV 1 μ L, CMV 1 μ L and 7.5 μ L ddH
2The O mixing, 70 ℃ of insulation 10min, rapid afterwards ice bath;
(b) reverse transcription: in aforementioned tube, add reagent by following system, the vibration mixing, room temperature is placed 10min, and 42 ℃ of insulation 1hr obtain cDNA;
(c) PCR: get a new PCR pipe, the according to the form below system adds each reagent:
The PCR program is set is: 94 ℃ of 4min, 94 ℃ of 1min, 56 ℃ of 1min, 72 ℃ of 2min, 26 circulations of increasing, 4 ℃ of preservations behind 72 ℃ of extension 10min;
4) electrophoresis detection: the Ago-Gel of preparation 0.9%, in 0.5 * tbe buffer pendular ring border, 150V voltage stabilizing electrophoresis 90min takes out gel afterwards and is immersed in the 10min that dyes in the 0.5 μ g/mL ethidium bromide, observes under uviol lamp then and the record result;
5) electrophoresis detection result: in the lily that has infected LMoV separately, detected size and be the nucleic acid band of 1153bp, illustrate by the present invention can be special detect LMoV; Detected size and be the nucleic acid band of 736bp in the lily that has infected LSV separately, illustrating can the special LSV of detecting by the present invention; Detected the nucleic acid band of 590bp in the lily that has infected CMV separately, illustrating can the special CMV of detecting by the present invention; Do not detect any band in the normal healthy controls, it is any to illustrate that this sample kind does not contain these three kinds of viruses.
Example three:
With known MOI the lily plant of LMoV and LSV, MOI the lily plant of LSV and CMV and MOI the lily plant of LMoV and CMV be material, with the negative contrast of healthy lily plant, detect with this method.
1) design primer:
(a) design detects the downstream universal primer LMSV of LMoV, two kinds of viruses of LSV:
LMSV 5-GATGGCTGACTGGATCCTTTTTTTTTTTTTTTTV-3 V=A, C or G
(b) design detects the upstream primer of LMoV, LSV, and primer sequence is as follows:
(c) design detects the upstream and downstream primer of CMV, and primer sequence is as follows:
(d) determine every pair of primer amplification clip size:
2) total RNA extracts:
(a) take by weighing lily blade 50mg, put into mortar, add 1mL Trizol reagent and grind, lapping liquid is poured in the 1.5mL centrifuge tube;
(b) add 400 μ L chloroforms, leave standstill 5min behind the mixing;
(c) 12, the centrifugal 10min of 000rpm;
(d) supernatant moves in the new 1.5mL centrifuge tube, adds isopyknic isopropyl alcohol, leaves standstill 5min behind the mixing;
(e) 12, the centrifugal 10min of 000rpm;
(f) abandon supernatant, precipitation is washed with 1mL 70% ethanol, dry 10min;
(g) precipitation is handled aqueous suspension with 40 μ L DEPC, and-20 ℃ of preservations are standby;
3)RT-PCR：
(a) RNA sex change: RNA 3 μ L, LMSV 1 μ L, CMV 1 μ L and 7.5 μ L ddH
2The O mixing, 70 ℃ of insulation 10min, rapid afterwards ice bath;
(b) reverse transcription: in aforementioned tube, add reagent by following system, the vibration mixing, room temperature is placed 10min, and 42 ℃ of insulation 1hr obtain cDNA;
(c) PCR: get a new PCR pipe, the according to the form below system adds each reagent:
The PCR program is set is: 94 ℃ of 4min, 94 ℃ of 1min, 57 ℃ of 1min, 72 ℃ of 2min, 28 circulations of increasing, 4 ℃ of preservations behind 72 ℃ of extension 10min;
4) electrophoresis detection: the Ago-Gel of preparation 1%, in 0.5 * tbe buffer pendular ring border, 150V voltage stabilizing electrophoresis 90min takes out gel afterwards and is immersed in the 10min that dyes in the 0.5 μ g/mL ethidium bromide, observes under uviol lamp then and the record result;
5) electrophoresis detection result: at MOI detected two nucleic acid bands of 1153bp and 736bp in the lily of LMoV and LSV, illustrate by the present invention and can synchronous detection go out LMoV and two kinds of viruses of LSV; In the blade that has infected LSV and two kinds of viruses of CMV, detected two nucleic acid bands of 736bp and 590bp, illustrated by the present invention and can synchronous detection go out LSV and two kinds of viruses of CMV; In the lily blade that has infected LMoV and two kinds of viruses of CMV, detected two nucleic acid bands of 1153bp and 590bp, illustrated by the present invention and can synchronous detection go out LMoV and two kinds of viruses of CMV; Do not detect any band in normal healthy controls, it is any to illustrate that this sample kind does not contain these three kinds of viruses.
Utilize the method under different situations, lily mottle virus (LMoV), lily asymptomatic virus (LSV) and cucumber mosaic virus (CMV) to be detected.Find out from experimental result, no matter be to infect separately in a kind of virus, still under two kinds or three kinds of compound situations about infecting of virus, all can in electrophoretogram, detect corresponding virus amplification product by ad-hoc location, and negative control do not have non-specific result and takes place, so this method can be applied to the LMoV, the LSV that take place on the lily plant and the synchronous detection of three kinds of main viruses of CMV.
Claims (4)
1, the method for a kind of synchronous detection LMoV, LSV and three kinds of viruses of CMV, its step is as follows:
1) design primer:
(a) design detects the downstream universal primer LMSV of LMoV, two kinds of viruses of LSV:
LMSV 5-GATGGCTGACTGGATCCTTTTTTTTTTTTTTTTV-3 V=A, C or G
(b) design detects the upstream primer of LMoV, LSV, and primer sequence is as follows:
(c) design detects the upstream and downstream primer of CMV, and primer sequence is as follows:
(d) determine every pair of primer amplification clip size:
2) total RNA extracts:
(a) take by weighing lily blade 50mg, put into mortar, add 1mL Trizol reagent and grind, lapping liquid is poured in the 1.5mL centrifuge tube;
(b) add 400 μ L chloroforms, leave standstill 5min behind the mixing;
(c) 12, the centrifugal 10min of 000rpm;
(d) supernatant moves in the new 1.5mL centrifuge tube, adds isopyknic isopropyl alcohol, leaves standstill 5min behind the mixing;
(e) 12, the centrifugal 10min of 000rpm;
(f) abandon supernatant, precipitation is washed with 1mL 70% ethanol, dry 10min;
(g) precipitation is handled aqueous suspension with 40 μ L DEPC, and-20 ℃ of preservations are standby;
3)RT-PCR：
(a) RNA sex change: RNA3 μ L, LMSV1 μ L, CMV1 μ L and 7.5 μ L ddH
2The O mixing, 70 ℃ of insulation 10min, rapid afterwards ice bath;
(b) reverse transcription: in aforementioned tube, add reagent by following system, the vibration mixing, room temperature is placed 10min, and 42 ℃ of insulation 1hr obtain cDNA;
(c) PCR: get a new PCR pipe, the according to the form below system adds each reagent:
The PCR program is set is: 94 ℃ of 4min, 94 ℃ of 1min, 53 ℃～60 ℃ 1min, 72 ℃ of 2min, 25～40 circulations of increasing, 4 ℃ of preservations behind 72 ℃ of extension 10min;
4) electrophoresis detection: the Ago-Gel of preparation 0.8%～2%, in 0.5 * tbe buffer pendular ring border, 150V voltage stabilizing electrophoresis 90min takes out gel afterwards and is immersed in the 10min that dyes in the 0.5 μ g/mL ethidium bromide, observes under uviol lamp then and the record result;
5) testing result analysis: having that it's too late size is determined the viral species that lily infects according to amplified band on the running gel; By observing, be the nucleic acid fragment of 1153bp if having size in the gel, then contain LMoV in the interpret sample; If having size in the gel is the nucleic acid fragment of 736bp, then contain LSV in the interpret sample; If having size in the gel is the nucleic acid band of 590bp, then contain CMV in the interpret sample.
2, used pcr amplification method in the method for synchronous detection LMoV according to claim 1, LSV and three kinds of viruses of CMV is characterized in that annealing temperature is 56 ℃～58 ℃.
3, used pcr amplification method in the method for synchronous detection LMoV according to claim 1, LSV and three kinds of viruses of CMV, 26～29 circulations is characterized in that increasing.
4, used electrophoretic detection in the method for synchronous detection LMoV according to claim 1, LSV and three kinds of viruses of CMV is characterized in that the concentration of the Ago-Gel prepared is 0.9%～1.2%.
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| CN101915835B (en) * | 2010-07-22 | 2013-06-19 | 中国科学院寒区旱区环境与工程研究所 | Lily mottle virus double-antibody sandwich enzyme-linked immunosorbent assay kit and preparation method |
| CN102367489B (en) * | 2011-10-13 | 2013-03-06 | 福建省农业科学院作物研究所 | Primer for detecting freesia mosaic virus (FreMV) and detection method of freesia mosaic virus |
| CN103243175B (en) * | 2013-05-06 | 2014-12-24 | 陈定虎 | Primers, kit and detection method for detecting lily symptomless virus |
| CN104122391B (en) * | 2014-04-14 | 2015-12-09 | 中国科学院寒区旱区环境与工程研究所 | A kind of hidden syndrome virus of lily and lily mottle virus composite colloid gold immunochromatographiassay assay reagent card and preparation method |
| CN105755173B (en) * | 2016-04-07 | 2019-10-25 | 中国科学院寒区旱区环境与工程研究所 | Three kinds of viral methods of RT-PCR synchronous detecting lily are captured with triple complex immunities |
| CN105695636A (en) * | 2016-04-07 | 2016-06-22 | 中国科学院寒区旱区环境与工程研究所 | Method for synchronously detecting lily symptomless virus (LSV), lily mottle virus (LMoV) and cucumber mosaic virus (CMV) |
| CN105803113B (en) * | 2016-04-07 | 2020-02-18 | 中国科学院寒区旱区环境与工程研究所 | Method for synchronously detecting lily latent virus and lily mottle virus by double composite immunocapture RT-PCR reaction |
| CN105713994B (en) * | 2016-04-07 | 2020-02-18 | 中国科学院寒区旱区环境与工程研究所 | Method for synchronously detecting lily latent virus and lily mottle virus by using double composite immunocapture RT-PCR (reverse transcription-polymerase chain reaction) |
| CN107164566B (en) * | 2017-07-19 | 2020-09-29 | 上海市农业科学院 | LAMP primer group, kit and detection method for detecting lily mottle virus |
| CN108967199B (en) * | 2018-09-28 | 2021-09-14 | 云南农业大学 | Detoxification and rapid propagation method for litsea cubeba |
| CN112458214B (en) * | 2020-12-28 | 2024-04-12 | 甘肃省农业科学院生物技术研究所 | Multiplex PCR primer for detecting lily virus and application of multiplex PCR primer in RNA extraction-free lily virus rapid detection method |
| CN114717359B (en) * | 2022-03-28 | 2023-10-27 | 华中农业大学 | Lily virus LCrV-1 specific detection target sequence, kit and detection method |
| CN114717360B (en) * | 2022-03-28 | 2024-04-26 | 华中农业大学 | Multiple RT-PCR detection kit and detection method for lily viruses |
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| CN1563411A (en) * | 2004-04-22 | 2005-01-12 | 南京农业大学 | Kit of testing garlic virus and testing method |
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| CN1485442A (en) * | 2002-09-24 | 2004-03-31 | 深圳市匹基生物工程股份有限公司 | Probe sequence for quantitatively detecting transgenic soybean containing cauliflower mosaic virus 35S promotor using fluorescence PCR and reagent case |
| CN1563411A (en) * | 2004-04-22 | 2005-01-12 | 南京农业大学 | Kit of testing garlic virus and testing method |
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